Project Summary Single-cell technologies have begun to reveal novel aspects of HIV-1 replication. However, it remains unclear how cellular heterogeneity influences transmissibility of virus-infected cells. We have developed quantitative single-cell assays for HIV-1 production and transmission. Our preliminary studies showed substantial cell-to- cell variations in both virus yield and transmissibility. Such diverse phenotypic outcomes of infection were unexpected and may offer new paradigms for HIV-1 biology. The major goal of this proposal is to gain quantitative and mechanistic insights into single-cell heterogeneity in HIV-1 production and transmission. In addition, a simple and rapid method for single-cell detection of virus-producing cells will be harnessed to develop novel assays for studying viral latency. In specific aim 1, we will study how virally induced diversity and cell-intrinsic heterogeneity contribute to cellular differences in productivity and infectiousness of individual virus-infected cells. A barcoded viral library will be utilized for high-throughput tracking of single-cell infection events in vitro and in an ex vivo tissue explant model. In specific aim 2, we will investigate how virus-infected cells shut off virion production during latency establishment to learn temporal changes in the cell fate during latency establishment. Single-cell detection of virion production will be used for quantification of the size of latent reservoirs and efficacy evaluation of latency reversal reagents. In specific aim 3, we will analyze single- cell transcriptome profiles to elucidate the biological basis for cell-to-cell heterogeneity of virus-infected cells by combining quantitative single-cell assays for virus production and transmission with single-cell RNA sequencing.